Heat-stable toxin (ST) produce by enterotoxigenic E. coli is a common cause of secretory diarrhea among children (in whom it can be fatal) and travels in areas where this pathogen is endemic. In mice, ST-induced diarrhea is prevented by ingestion of human milk. This protective activity has been localized to human milk fucosylooligosaccharides that have alpha 1, 2 linkages at their reducing ends. The amount of alpha 1, 2-linked to other fucosylooligosaccharides in mothers' milk is inversely related to the incidence of ST-associated diarrhea in their breastfeeding infants. We conclude that 2-linked fucosyl moieties are critical determinants in protection against ST, probably working in concert with 3- and 4-linked moieties. The fucosyltransferases that synthesize these fucosylooligosaccharides are thought to be expressed by genes of the secretor and Lewis family. We will synthesize a complete combinatorial library of fucosylated tri- and tetrasccharides encompassing the possible products of human milk fucosyltransferases. And then test their ability individually and in combinations, to inhibit ST-induced diarrhea in suckling mice. The activities of the most promising compounds will be compared with that of the fucosyloligosaccharide fraction of human milk. The mechanism of inhibition will be studied in cultured human enterocytes. The genetics basis for different levels of protective oligosaccharide in milk from different individuals will be studied. This project will identify fucose. The project will identify fucose-containing structures that inhibit ST, their relative efficacies, and their mechanisms of action. The genetic basis for the expression of fucosylooligosaccharides in milk will be determined. The relationship between their presence in milk and risk of ST diarrhea in infants will be investigated. The long-range goals are to develop strategies for determining which infants are at highest risk of this often deadly disease and produce novel, therapeutic fucosylooligosaccharides as a complement for infants at risk.